Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp E79 and the DNA sequence of the encoding gene

被引:30
作者
Lee, JK
Kim, YO
Kim, HK
Park, YS
Oh, TK
机构
[1] KIST, KOREA RES INST BIOSCI & BIOTECHNOL, APPL MICROBIOL RES GRP, TAEJON 305600, SOUTH KOREA
[2] KYUNGWON UNIV, DIV CHEM & FOOD ENGN, SUNGNAM 461701, SOUTH KOREA
关键词
thermostable alkaline protease; Thermoactinomyces sp; protein purification; DNA sequence; amino acid sequence similarity;
D O I
10.1271/bbb.60.840
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil, The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31,000 by SDS-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease, The optimum temperature for the enzyme activity was 85 degrees C, and about 50% of the original activity remained after incubation at 90 degrees C for 10 min in the presence of Ca2+. The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment, Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a polypeptide of 384 amino acids, The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids, The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.
引用
收藏
页码:840 / 846
页数:7
相关论文
共 32 条
[1]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[2]   THERMOPHILIC PROTEASES - PROPERTIES AND POTENTIAL APPLICATIONS [J].
COWAN, D ;
DANIEL, R ;
MORGAN, H .
TRENDS IN BIOTECHNOLOGY, 1985, 3 (03) :68-72
[3]   PURIFICATION AND PROPERTIES OF THE HIGHLY THERMOSTABLE ALKALINE PROTEASE FROM AN ALKALIPHILIC AND THERMOPHILIC BACILLUS SP [J].
FUJIWARA, N ;
MASUI, A ;
IMANAKA, T .
JOURNAL OF BIOTECHNOLOGY, 1993, 30 (02) :245-256
[4]   PRODUCTION OF ALKALINE ENZYMES BY ALKALOPHILIC MICROORGANISMS .1. ALKALINE PROTEASE PRODUCED BY BACILLUS N-2210 [J].
HORIKOSHI, K .
AGRICULTURAL AND BIOLOGICAL CHEMISTRY, 1971, 35 (09) :1407-+
[5]   SIGNAL PEPTIDES - EXQUISITELY DESIGNED TRANSPORT PROMOTERS [J].
IZARD, JW ;
KENDALL, DA .
MOLECULAR MICROBIOLOGY, 1994, 13 (05) :765-773
[6]   CLONING, SEQUENCING AND EXPRESSION OF SUBTILISIN CARLSBERG FROM BACILLUS-LICHENIFORMIS [J].
JACOBS, M ;
ELIASSON, M ;
UHLEN, M ;
FLOCK, JI .
NUCLEIC ACIDS RESEARCH, 1985, 13 (24) :8913-8926
[7]  
KLEINE R, 1982, ACTA BIOL MED GER, V41, P89
[8]  
KOMAGATA K, 1987, METHOD MICROBIOLOGY, V19, P160
[9]   HIGHLY THERMOSTABLE NEUTRAL PROTEASE FROM BACILLUS-STEAROTHERMOPHILUS [J].
KUBO, M ;
MURAYAMA, K ;
SETO, K ;
IMANAKA, T .
JOURNAL OF FERMENTATION TECHNOLOGY, 1988, 66 (01) :13-17
[10]  
LACEY J, 1989, BERGEYS MANUAL SYSTE, V4, P2573